Synthetic Glucocorticoids in the Environment - American Chemical

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Synthetic Glucocorticoids in the Environment: First Results on Their Potential Impacts on Fish Subramaniam Kugathas* and John P. Sumpter Institute for the Environment, Brunel University, Uxbridge, Middlesex UB8 3PH U.K. ABSTRACT: Human pharmaceuticals have been shown to be entering the aquatic environment in quantities that may produce adverse effects to aquatic organisms. This paper investigates the impacts of synthetic glucocorticoids (GCs), which are used in large amounts as anti-inflammatory drugs, on fish. Mammalian cell lines were transiently transfected with trout corticosteroid receptors (GR1, GR2, and MR) and the transactivation abilities of ten of the most prescribed GCs in the UK were measured in vitro. They showed significantly higher activity with GR2 than with GR1. In order to assess any impacts in vivo, adult fathead minnows were exposed to either 1 μg prednisolone/L or 1 μg beclomethasone dipropionate/L for 21 days. Plasma glucose concentrations were increased and leucocytes were reduced significantly in GC-exposed groups compared to the control group. In another experiment, fish were exposed to three different concentrations of Beclomethasone dipropionate and a dose-dependent increase of plasma glucose was found. The results suggest that low concentrations of synthetic GCs present in water could cause adverse effects on fish. Therefore, quantification of GCs in the aquatic environment and the effects of GCs at environmentally relevant concentrations are required in order to determine if GCs pose a threat to wild fish populations.

’ INTRODUCTION Pharmaceuticals and personal care products have been detected in many environmental samples, such as sewage treatment plant effluents, surface water, groundwater, sediments, and fish, worldwide.1-3 It has also been reported that drug manufacturing site effluents4 and hospital waste waters5 can contain high levels of human pharmaceuticals and their metabolites which may have adverse effects on living organisms.6 For example, ibuprofen7 and diclofenac8 have been reported to have acute toxicity to aquatic organisms at environmentally relevant concentrations, and residues of diclofenac are the cause of the almost complete loss of the populations of three species of vultures in the Indian subcontinent.9 However, it is important to emphasize that currently it is not possible to know whether or not the concentrations of pharmaceuticals present in the aquatic environment are adversely affecting aquatic organisms. This is because reliable measurements of concentrations of pharmaceuticals in rivers are very sparse and because few of the reported effects of pharmaceuticals on aquatic organisms have been independently repeated. A steroid pharmaceutical, ethinyl estradiol (EE2), has been extensively studied (reviewed in ref 10) and concentrations below 1 ng/L have adverse effects on fish reproduction.10,11 However, none of the other classes of steroidal pharmaceuticals have received much attention. Given the ease with which steroids can enter fish from the environment,12 this represents a significant knowledge gap. The recent demonstration that synthetic progestogens can adversely affect fish at very low concentrations13,14 has confirmed the need to closely evaluate the environmental effects of steroidal pharmaceuticals. Currently nothing is known about the impacts of synthetic GCs present in the aquatic environment. GCs are used in treating a variety of diseases, including asthma, rheumatic disease, inflammatory bowel disease, inflar 2011 American Chemical Society

mmatory skin, and eye and ear conditions. The average daily dose of corticosteroids varies from 100 μg to 500 mg, depending on the preparation and the route of administration.15 In many of the synthetic corticosteroids, H on the ninth carbon is substituted by a halogen (Table 1). This substitution increases the stability of these compounds in the human body. Therefore it is anticipated that halogenated corticosteroids will biodegrade relatively slowly, and hence could be found in the aquatic environment. Synthetic corticosteroids in surface waters and effluents16,17 have been detected in ng/L concentrations. In fish, the interrenal cells secrete corticosteroid hormones, including cortisol and corticosterone, which modulate functions such as glucose metabolism, stress response, regulation of blood pressure, and osmoregulation.18 Fish lack aldosterone, and cortisol has been shown to have both gluco and mineralocorticoid activity.19 The effects of corticosteroid hormones are mediated through intracellular receptors that act as liganddependent transcription factors. In teleost fish, three corticosteroid receptors are present: two glucocorticoid receptors (GR1 and GR220,21) and a mineralocorticoid receptor (MR 19) have been sequenced and reported. In vivo studies on synthetic corticosteroids and fish are very rare. Most studies so far have applied treatments via implanted capsules or food, and involved high concentrations of GC compared to the expected environmentally relevant concentrations.22,23 No studies have been reported using the environmentally relevant exposure route, via the water. The present study is the first report of the effects of chronic exposure of fish to low concentrations of synthetic GCs. Received: October 5, 2010 Accepted: January 19, 2011 Revised: January 17, 2011 Published: February 15, 2011 2377

dx.doi.org/10.1021/es104105e | Environ. Sci. Technol. 2011, 45, 2377–2383

Environmental Science & Technology

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Table 1. Chemical Structures, logP Values, and the Amounts Prescribed for the Ten Most Prescribed GCs in the UK in 2006

GC

C1, 2

C6

C9

hydrocortisone CAS 50-23-7 prednisolone CAS 50-24-8

a

=

C11

C16

C17

LogPa

annual amount prescribed (kg)24

-OH

HOCH2C = O, -OH

1.79

1810.91

-OH

HOCH2C = O, -OH

1.66

1488.30

betamethasone CAS 378-44-9

=

F

-OH

-CH3

HOCH2C = O, -OH

1.93

305.33

beclometasone CAS 4419-39-0 fluticasone CAS 90566-53-3

= =

Cl F

-OH -OH

-CH3 -CH3

HOCH2C = O, -OH F-CH2SC=O, -OH

2.12 2.69

273.96 176.29

budesonide CAS 51333-22-3

=

-OH

-OCHCH2CH3

HOCH2C = O, -O-

2.42

89.46

momethasone CAS 105102-22-5

=

Cl

-OH

-CH3

ClCH2C = O, -OH

2.81

51.81

=O

-CH3

clobetasone CAS 54063-32-0

=

methylprednisolone CAS 83-43-2

=

dexamethasone CAS 50-02-2

=

-F

F -CH3

-OH F

-OH

-CH3

ClCH2C = O, -OH

2.88

44.72

HOCH2C = O, -OH

2.06

28.98

HOCH2C = O, -OH

1.93

27.26

Estimated Log P value (by ALOGPs: www.vcclab.org).

’ MATERIALS AND METHODS In Vitro Assessment. The total amount of synthetic corticosteroids used in the UK was calculated as previously described.24 The ten GCs prescribed in the highest amounts were chosen for study, and their potencies/efficacies with GR and MR were studied in vitro. Propagation of Plasmid DNA. Receptor expression vector (pCMrtGR2, 50 ng) was transformed with 100 μL of E-coli strain (J109, Promega) by the heat shock method and propagated in super optimal broth with catobolite repression. Different dilutions of the above culture were spread on lysogeny broth (LB) agar plates together with ampicillin. After overnight incubation at 37 °C, isolated colonies were transferred to 5 mL of LB for shaking incubation overnight. A 300 μL portion of this culture was added to 150 mL of LB the next day and incubated overnight. Receptor plasmid DNA was extracted using Hi speed plasmid purification kits (Qiagen) according to the manufacturer’s instructions. Purity of the plasmid DNA was confirmed with agarose gel electrophorosis and the concentration was measured spetrophotometrically (Nanodrop) in ng/μL. The same procedure was repeated for pFC31Luc, which contains the mouse mammary tumor virus promoter upstream of the luciferase gene, and pSVβ containing the gene coding for the βgalactosidase enzyme. Transfection Assay. Mammalian cell lines (COS-7) were grown in Dulbecco’s Modified Eagle Medium (DMEM-Invitrogen) supplemented with 100 IU/mL penicillin, 100 μg/ mL streptomycin (Sigma), 2 mM glutamine (Sigma), and 10% denatured fetal calf serum (Invitrogen) in a humidified atmosphere (37 °C) with 5% CO2. For recovery from liquid nitrogen

storage, cells were propagated two times a week for two weeks in 75 cm3 flasks (Fisher, UK) with 20 mL of medium inoculated with 0.5 million cells. For the transfection assay, 20 000 cells were inoculated in 1 mL of medium in each well of 12 well plates. Four hours before transfection and throughout the rest of the experiment, cells were maintained in DMEM nutrient mixture F-12 Ham (Sigma) supplemented with 100 IU/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, 3.7 g/liter NaHCO3, and 2.5% desteroided denatured fetal calf serum (Sigma). Cells were transiently transfected by the Gene juice transfection reagent (Novagen). The transfection mixture, prepared for a 12-well plate, contained 2 μg of the receptor expression vector (pCMrtGR2), 4 μg of pFC31Luc, and 1 μg of pSVβ. Twelve hours after transfection, the medium was renewed and the GCs (Sigma) were added from 1000-fold concentrated stock solutions in ethanol. After 36 h incubation, cells were harvested with lysis buffer (Promega) and extracts were analyzed for luciferase (Promega) and β-galactosidase activities. Solvent-controls with triplicate cell cultures per treatment were included in each assay. For the luciferase assay, 10 μL of cell extract was placed in a well of a 96-well plate and read by the luminometer (Glo max, Promega) with settings of 10 s reading and 2 s delay time using single injector for addition of 50 μL of reagent buffer to each well, so that the luminometer could perform readings immediately after the addition of reagent. This ensured loss of activity was prevented. For the β-Galactosidase assay, KP buffer at pH 7.3 was prepared with K2HPO4 3 3H2O and KH2PO4 (Fisher, UK). Ortho-nitrophenyl β-galactopyranoside (ONGP) and 2-mercaptoethanol were purchased from Sigma. For a 96-well plate, 2378

dx.doi.org/10.1021/es104105e |Environ. Sci. Technol. 2011, 45, 2377–2383

Environmental Science & Technology 13.7 mL of KP buffer, 240 μL of MgCl2/2-mercaptoethanol and 5.3 mL of ONGP stock were mixed. A 200 μL portion of this mixture was added to 50 μL of cell extract in clear 96-well plates and incubated in 37 °C. After yellow color formation, reaction time was noted and the plate was read by the plate reader (Spectramax 340PC) at 420 nm. Luciferase activity was corrected for well-specific transfection efficiency (as determined by β-galactosidase activity) and then expressed as percentage transcriptional activity, taking the luciferase activity of 10-6 M hydrocortisone as 100%. Third-order polynomial curves obtained with Excel were optimized with datafit software (version 9: Oakdale Engineering, www.oakdaleengr. com) in order to calculate the EC50 (concentration of hormone to produce half maximal activity) values. In Vivo Assessment. To assess the impact of low concentrations of GCs in vivo, two exposure experiments were conducted with adult fathead minnows, in 30-L glass tanks, using a continuous flow-though system. Fish were selected from breeding stock maintained at Brunel University, and fed three times per day, once with adult brine shrimp (Tropical Marine Centre, Gamma irradiated) and twice with flake food (King British Tropical flake food, Surrey). In the first experiment, one group of 10 fish was exposed to 1 μg of prednisolone/L, another group to 1 μg of beclomethasone dipropionate/L, and the third group served as control. Prednisolone (CAS 50-24-8, 99% purity, Sigma-Aldrich, UK) and beclomethasone dipropionate (CAS 5534-09-8, 99% purity, Sigma-Aldrich, UK) were dissolved in ethanol, and stock solutions (1 mg/L) were freshly prepared in 2.5-L amber bottles every fifth day, by dissolving the GC in double-distilled water and stirring vigorously overnight. Stock solutions were dosed at 18 mL/h, using a Watson Marlow (Cornwall, UK) multichannel peristaltic pump, into glass mixing vessels (aspirator bottles), where they mixed with dechlorinated tap water (at 18 L/h) before delivery to each fish tank to produce the desired concentrations. Flow rates and dosing efficiency were monitored daily to ensure that GC entered the fish tanks at the expected rates. Water samples (500 mL) were taken 3 times on the days when the stock solutions were changed and kept frozen for analysis. All tubing within the system was medical grade silicon. Dosing of the tanks with GCs was carried out for a week prior to fish being put into the tanks, to allow the system to equilibrate. During this equilibration period, fish were acclimatized in similar experimental conditions. Temperature (25.2 ( 1.02 °C) and dissolved oxygen (7.12 ( 0.92 mg/L) were monitored daily throughout the experiment. After 21 days of exposure, fish were terminally anesthetized using MS-222 (Sigma, Poole, UK). During the procedure, fish were treated humanely with regard to alleviating suffering. Weight in grams and fork length in mm were measured. The tail was then removed from each fish and blood was withdrawn using 75-μL capillary tubes, then decanted into eppendorf tubes (on ice). The cut edge of the tail was used to make a blood film on a microscopic slide, for staining and differential cell counting. Fresh blood (25 μL) was transferred into 1975 μL of NattHerrick’s stain solution in an eppendorf tube for total cell count using a hemocytometer. Fish were dissected to obtain the liver and gonad weights. Condition factor, liver-somatic index, and gonadosomatic index were calculated. Blood samples were kept on ice until plasma was separated by centrifugation for 4 min at 14 000g, and this was then stored at -20 °C for glucose measurement. Blood glucose was determined using a

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quantichrome kit (Universal biological, UK). Leukocyte counts were performed based on the method described in ref 25. Prednisolone concentrations in tank water samples were measured with Enzyme Immunoassay Kit (Cambridge Bioscience, Cat. no. 900-071), according to manufacturer’s instructions. Beclomethasone dipropionate concentrations in water samples were not measured. In the second experiment, fish (n = 20) were exposed to 3 different concentrations (100 ng/L, 1 μg/L, and 10 μg/L) of beclomethasone dipropionate for 21 days. Flow-through conditions and the experimental set up were similar to the first experiment. At the end of exposure, plasma glucose levels were measured as described above. Statistical Analyses. Experimental results are presented as means ( standard errors. As appropriate, normality was determined and statistical significances were tested with a t test or ANOVA using SigmaStat 3.5. p < 0.05 was considered to be significant. For the dose-response effect in the second experiment, in addition to ANOVA, trend analysis (using SPSS: Jonckheere-Terpstra non parametric test) was done.

’ RESULTS Figure 1 reveals the relative potencies of the top 10 most used corticosteroids with trout GR2. As in mammals, these ten GCs could be classified as low, medium, and high potency. Trout GR1 also responded to all the corticosteroids, but with lower transcriptional activity (efficacy). Figure 2 compares the transcriptional activities (as fold of solvent control) of tested GCs at 1 μM concentrations with GR1, GR2, and MR. Overall, there is approximately an 8-12-fold difference in the sensitivity (as efficacy) of GCs between GR2 and GR1. None of the tested GCs produced significant activity with fish MR. EC50 values of each GC with GR1 and GR2 are presented in Table 2. The results of the in vivo experiments are shown in Figures 3, 4, and 5. In experiment 1, plasma glucose concentrations were significantly (one way ANOVA; p < 0.001) increased in GC exposed groups of fish (79.56 ( 10.39 mg/dL and 87.39 ( 11.39 mg/dL for prednisolone and beclomethasone dipropionate, respectively) compared to the control group (54.42 ( 8.07 mg/dL). The effect of beclomethasone was higher than that of prednisolone, but the difference was not significant. The anti-inflammatory properties of GCs were also revealed, as the total leucocyte count was reduced significantly by both prednisolone and beclomethasone. Total leucocytes were reduced significantly(one way ANOVA; p < 0.001) in GC-exposed groups (126.1 ( 8.99 103 mm-3 and 119.1 ( 7.21 103 mm-3 for prednisolone and beclomethasone, respectively) compared to the control group (144.7 ( 10.83 103 mm-3). There were no differences in the condition factor, liver-somatic index, or gonadosomatic index between control and treated groups (data not shown). In experiment 2, there was a dose-related increase of plasma glucose levels in beclomethasone dipropionate - treated fish. Glucose levels in fish exposed to 1 and 10 μg/L were significantly higher than that of control fish (Figure 5). Measured prednisolone concentrations in tank water were not significantly different between sampling days (Figure 6). Average concentration of all 15 samples was 902.5 ( 124.6 ng/L, compared to the nominal concentration of 1000 ng/L. Prednisolone concentrations in the control tanks were below detection limit (less than 56 pg/mL). 2379



’ DISCUSSION GCs were not included in previous UK based studies involving environmental risk assessment of human pharmaceuticals.26,27 The above studies revealed that many of the highly consumed pharmaceutical substances, such as paracetamol and aspirin, are readily biodegradable. In contrast, the impacts of EE2 in the environments have been well documented (reviewed in ref 10) and the annual usage of EE2 in the UK (about 25 kg) is well below that of most of the GCs used in this study.

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There are approximately 30 different GCs currently licensed for use in the UK.15 To prioritize those GCs most likely to be present at measurable concentrations in the UK environment, and hence those that may present a risk to fish, it is important to establish which GCs are used to the greatest extent in the UK. Statistics on how much is spent on different pharmaceuticals by the National Health Service have been produced by the Department of Health. Though this database does not cover private hospital prescriptions, it is estimated that it accounts for about 80% of use in the UK. Thus, this database probably provides a reliable assessment of the amounts of the different GCs used annually in the UK.24 The most-used GC in the UK, hydrocortisone, is readily degradable. The second most used GC, prednisolone, has been reported16 to be the most frequently detected GC in effluent in China. This in turn was found to be a result of its relatively low efficiency of biodegradation. Although other GCs are used in relatively low amounts compared to prednisolone, their structural modifications, designed to make them more stable in patients, could mean that they are present in the environment at concentrations higher than might otherwise be expected. Table 2. EC50 Values of Tested GCs with Trout GR2 and GR1 corticosteroid

Figure 1. Percentage luciferase activity (normalized against β-galactosidase activity for the transfection efficiency) of ten GCs in trout GR2 transfected COS-7 cells. Luciferase activity of 10-6 M hydrocortisone was considered as 100% activity. Values are the average of three replicates.

EC50 with trout GR2 (nM) EC50 with trout GR1 (nM)

dexamethasone beclomethasone

0.386 ( 0.02 0.737 ( 0.07

40.98 ( 2.43 44.78 ( 1.98

betamethasone

0.771 ( 0.09

42.34 ( 2.78

fluticosone

0.805 ( 0.02

45.09 ( 4.56

mometasone

0.816 ( 0.01

47.24 ( 3.90

budisonide

0.856 ( 0.11

52.64 ( 6.32

prednisolone

1.12 ( 0.03

52.09 ( 7.98

hydrocortisone

1.22 ( 0.12

50.21 ( 2.54

clobetasone

1.86 ( 0.91

61.09 ( 9.02

a

Values are the mean and standard deviation of three independent experiments performed in duplicate.

Figure 2. Transcriptional activities (as measured by luciferase activity normalized to β-galactosidase activity and presented as fold of activity of the solvent control) of ten highly consumed GCs in UK, measured at 1 μM concentrations, with trout GR1, GR2, and MR. Values are the mean of three replicates and the standard deviation. 2380



Figure 3. Plasma glucose concentrations in control and treated (either 1 μg prednisolone/L or 1 μg Beclomethasone dipropionate/L) groups of fish. (n = 10, mixed sex in each group). * indicates significant difference (ANOVA: p < 0.001).

Figure 4. Blood leukocyte counts in control and treated (either 1 μg prednisolone/L or 1 μg beclomethasone dipropionate/L) groups of fish. (n = 10, mixed sex in each group). * indicates significant difference (ANOVA: p < 0.001).

Figure 5. Plasma glucose concentrations in untreated (control) fish, and in fish exposed to low (100 ng/L), medium (1 μg/L), and high (10 μg/L) concentrations of beclomethasone dipropionate. * indicates the significant difference from control (p < 0.05 in one way ANOVA followed by tukey pair wise comparison). There was a highly statistically significant concentration-related trend (trend analysis using SPSS: Jonckheere-Terpstra (p < 0.0001)).

Mammalian cell lines transiently transfected with fish GR and MR have been shown to respond to natural and some synthetic

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corticosteroids.19 Specifically, cortisol binds to and induces the transcriptional activity of both GR and MR. Therefore this transfection assay is a useful tool to compare the activity of different synthetic corticosteroids in fish. Strum et al.19 have also reported higher luciferase activity for trout GR2 than for GR1, as occurred in our study. EC50 values of hydrocortisone for GR2 and GR1 fell within the range previously reported.19 The present study is the first to report the comparison of the potencies of a significant number of the synthetic GCs used in the UK. The potencies of these GCs in humans15 are in an order similar to that reported for the fish GR in the present study. Results indicate that GCs bind to fish GR in a manner similar to that reported for mammalian receptors. These relative potencies of different GCs can be used to predict the effect of a mixture of GCs, as might occur in the environment, similar to the way that the effects of mixtures of estrogens have been predicted.28 It is well established that GCs induce a significant tendency to hyperglycemia in humans. Gluconeogenesis, a major effect of GCs on glucose metabolism, has been well documented.29 This effect of GCs also occurs in fish. Cortisol administration to fish elevated the plasma glucose concentration 2- to 3-fold, depending on the concentration.30 The timing of peak plasma glucose levels following stress coincided with the timing of elevated cortisol levels, indicating a role of cortisol in mobilizing glucose during stress in teleosts.18 Based on the effect of natural corticosteroids on plasma glucose concentrations, we hypothesized that synthetic GCs might also cause an elevation in the plasma glucose concentration. Both synthetic GCs tested caused an increase in the plasma glucose concentration of about 50%. Future research should establish concentration relationships, thus enabling the determination of the lowest observed effect concentration (LOEC), no observed effect concentration (NOEC), and the predicted no effect concentration (PNEC). These values are important in assessing the risks of such chemicals to the environment. A number of human trials have revealed the reduction of leukocyte counts after treatment with synthetic GCs. Similarly, dexamethasone treatment in fish significantly reduced the leukocyte count.22 Lymphocytopenia is a consequence of acute and chronic stress in trout,25,31 and is a direct effect of the elevation of plasma cortisol levels. Since the previous studies induced stress effects by either implantation of cortisol, handling and confinement, or cortisol administration via food, it is difficult to compare them directly with the present study. It may also be relevant that cortisol is a natural GC in fish and its concentration is elevated in response to stress, but it is less potent than the GCs used in the present exposure studies. Several pharmaceuticals widely used in human medicine are excreted unchanged or as active metabolites in high percentages, and hence are continuously discharged into domestic waste waters.5 Unwanted or expired medications are often improperly disposed of directly to wastewater. Furthermore, many GCs are used in topical creams, which are then washed off into the sewage system and could reach STPs in substantial amounts. Use of GCs for farm animals may also contribute to the exposure.17 Most wastewater treatment plants do not completely remove therapeutic compounds, which subsequently flow into surface waters, and can eventually reach ground waters.1 The results demonstrate that relatively low concentrations of synthetic GC (1 μg/L) can cause effects on fish. The second experiment confirmed the results of the first experiment, demonstrating their repeatability, and also showed that effects are 2381



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’ ACKNOWLEDGMENT We thank Dr. Nick Bury and Dr. Armin Strum for providing the receptor plasmids, the Fish Biology Group of Brunel University for help in the exposure studies, and the Commonwealth Commission for financial support of S.K. ’ REFERENCES

Figure 6. Concentrations of prednisolone in tank water of Experiment 1, sampled on day 0, 5, 10, 15, and 21. Fish were transferred into experimental tank on day 0 but dosing the tanks was started one week before day 0. Values are the average and standard deviation of triplicate samples per day.

concentration-related, with higher concentrations causing effects of greater magnitude. We are not currently able to define a NOEC for this chemical, because all three concentrations tested caused an increase in the plasma glucose concentrations. However, exposure to 100 ng/L did not cause a statistically significant increase, therefore it could be argued that the NOEC is 100 ng/L, and hence the LOEC is 1 μg/L. These need refinement in subsequent experiments. The results suggest that GCs could cause effects at very low (as low as 100 ng/L) concentrations that could be environmentally relevant. These effects are potentially important. For example, lymphocytopenia in fish is associated with increased susceptibility to disease.31 The next logical step in risk assessment of these chemicals would be to establish concentration-response relationships, building on the one shown here, and to compare these with reported environmental concentrations of GCs, in order to ascertain whether or not environmental concentrations of GCs are high enough to cause immunosuppression in wild fish. If they are, then the exposed fish would likely be more susceptible to disease. Because many different GCs are in widespread clinical use, it seems likely that, concurrently, many different GCs will be present in the aquatic environment. As our binding studies demonstrate, all of the GCs tested here can bind to the fish GR (as expected), and therefore it can be argued that the total concentration of GC in the environment, rather than the concentration of each individual GC, is of most relevance to the risk assessment of GCs on aquatic organisms. However, currently not enough is known to provide a full picture of GCs in the environments. Nevertheless, it is very likely that the effects of different GCs will be additive, as has been shown for estrogenic chemicals.28 Therefore, research needs to be carried out to quantify the concentrations of these pharmaceutical substances in STP effluent and in surface water, and ecotoxicological research conducted at these concentrations. Thus there are many issues to be resolved before a robust environmental risk assessment of GCs can be completed.

’ AUTHOR INFORMATION Corresponding Author

*Tel: þ44 1895 274000; fax: þ44 1895 269761; e-mail: [email protected].

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